*Target (protein/gene name): UDP-N-acetylenolpyruvylglucosamine reductase *NCBI Gene # or RefSeq#:931443 *Locus Tag: murB *Protein ID (NP or XP #) or Wolbachia#: YP_816689.1 *Organism (including strain):Streptococcus pneumoniae Etiologic Risk Group (see link below): Risk Group 2 *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Essentiality of this protein: Is essential. Complex of proteins?: No Druggable Target: Yes " The enzymes involved in peptidoglycan biosynthesis are among the best-known targets in the search for new antibiotics" - Yang, et. al.
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1MBT
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB Top: UDP-N-acetylenolpyruvylglucosamine reductase in S. pneumoniae Bottom: UDP-N-acetylenolpyruvylglucosamine reductase in E. coli
---- Query Coverage: 94%
---- Max % Identities:52%
---- % Positives: 68%
---- Chain used for homology: Chain A
Current Inhibitors:3,5-Dioxopyrazolidines Expression Information (has it been expressed in bacterial cells): Yes Purification Method: ADP-Sepharose column elution purification [Details] Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSVREKMLEILEGIDIRFKEPLHSYSYTKVGGEADYLVFPRNRFELARVVKFANQENIPWMVLGNASNII
VRDGGIRGFVILCDKLNNVSVDGYTIEAEAGANLIETTRIALRHSLTGFEFACGIPGSVGGAVFMNAGAY
GGEIAHILQSCKVLTKDGEIETLSAKDLAFGYRHSAIQESGAVVLSVKFALAPGTHQVIKQEMDRLTHLR
ELKQPLEYPSCGSVFKRPVGHFAGQLISEAGLKGYRIGGVEVSEKHAGFMINVADGTAKDYEDLIQSVIE
KVKEHSGITLEREVRILGESLSVAKMYAGGFTPCKR
*length of your protein in Amino Acids: 316 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 34.5387kDa Molar Extinction coefficient of your protein at 280 nm wavelength:
6,220 cm^-1M^-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
TMpred indicates that the protein has a high likelihood of being transmembrane. However, empirical studies
indicate that the protein is not transmembrane, but globular and the Philius prediction model concurs.
*GC% Content for gene: 45% [Found Using Genomics Content Calculator] *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
*NCBI Gene # or RefSeq#: 931443
*Locus Tag: murB
*Protein ID (NP or XP #) or Wolbachia#: YP_816689.1
*Organism (including strain): Streptococcus pneumoniae
Etiologic Risk Group (see link below): Risk Group 2
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Essentiality of this protein: Is essential.
Complex of proteins?: No
Druggable Target: Yes "
The enzymes involved in peptidoglycan biosynthesis are among the best-known targets in the search for new antibiotics" - Yang, et. al.
*EC#: 1.1.1.158
Link to BRENDA EC# page: http://brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.158
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Tris-HCl (pH 8.0), 10 mM KCl, 100µM NADPH, and 50µM enolpyruvyl-UDP-N-acetylglucosamine(EP-UNAG)
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
NADPH
EP-UNAG
-- link (or citation) to paper that contains assay information
Yang, Y.; Severin, A.; Chopra, R.; Krishnamurthy, G.; Sing, G.; Hu, W., Keeney.; Svenson, K.; Petersen, P.; Labthavikul, P.; Shales, D.; Rasmussen, B.; Failli, A.; Shumsky, J.; Kutterer, K.; Gilbert, A.; Mansour, T., 3,5-Dioxopyrazolidines, Novel Inhibitors of Acetylenolpyruvylglucosamine-N-UDP Reductase (MurB) with Activity against Gram-Positive Bacteria. Antimicrob. Agents Chemother. 2006, 50(2), 556.
-- List cost and quantity of substrate reagents and supplier
NADPH: $66.20 per 25mg
EP-UNAG: $51.40 per 25mg
EP-UNAG was synthesized and purified according to the published method - Yang, et. al.
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1MBT
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Top: UDP-N-acetylenolpyruvylglucosamine reductase in S. pneumoniae
Bottom: UDP-N-acetylenolpyruvylglucosamine reductase in E. coli
---- Query Coverage: 94%
---- Max % Identities:52%
---- % Positives: 68%
---- Chain used for homology: Chain A
Current Inhibitors: 3,5-Dioxopyrazolidines
Expression Information (has it been expressed in bacterial cells): Yes
Purification Method: ADP-Sepharose column elution purification [Details]
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSVREKMLEILEGIDIRFKEPLHSYSYTKVGGEADYLVFPRNRFELARVVKFANQENIPWMVLGNASNII
VRDGGIRGFVILCDKLNNVSVDGYTIEAEAGANLIETTRIALRHSLTGFEFACGIPGSVGGAVFMNAGAY
GGEIAHILQSCKVLTKDGEIETLSAKDLAFGYRHSAIQESGAVVLSVKFALAPGTHQVIKQEMDRLTHLR
ELKQPLEYPSCGSVFKRPVGHFAGQLISEAGLKGYRIGGVEVSEKHAGFMINVADGTAKDYEDLIQSVIE
KVKEHSGITLEREVRILGESLSVAKMYAGGFTPCKR
*length of your protein in Amino Acids: 316
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 34.5387kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
6,220 cm^-1M^-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
TMpred indicates that the protein has a high likelihood of being transmembrane. However, empirical studies
indicate that the protein is not transmembrane, but globular and the Philius prediction model concurs.
*CDS Gene Sequence (paste as text only):
ATGTCTGTAAGAGAAAAAATGCTTGAAATCTTAGAAGGAATTGATATCCGTTTTAAGGAACCCTTGCATA
GCTATAGTTATACAAAAGTAGGTGGAGAGGCTGATTATTTGGTCTTTCCACGAAATCGTTTTGAGTTGGC
TCGCGTTGTGAAATTTGCCAACCAAGAAAATATCCCTTGGATGGTTCTTGGCAATGCAAGCAATATCATC
GTTCGTGATGGTGGGATTCGTGGATTTGTCATCTTGTGTGACAAGCTCAATAACGTTTCTGTTGATGGCT
ATACCATTGAAGCAGAAGCTGGGGCTAACTTGATTGAAACAACTCGCATTGCCCTCCGTCATAGTTTAAC
TGGCTTTGAGTTTGCTTGTGGTATTCCAGGAAGCGTTGGCGGTGCTGTCTTTATGAATGCGGGTGCCTAT
GGTGGCGAGATTGCTCACATCTTGCAGTCTTGTAAGGTCTTGACCAAGGATGGAGAAATCGAAACCCTGT
CTGCTAAAGACTTGGCTTTTGGTTACCGTCATTCAGCTATTCAGGAGTCTGGTGCAGTTGTCTTGTCAGT
TAAATTTGCCCTAGCTCCAGGAACCCATCAGGTTATCAAGCAGGAAATGGACCGCTTGACGCACCTACGT
GAACTCAAGCAACCTTTGGAATACCCATCTTGTGGCTCGGTCTTTAAGCGTCCAGTCGGGCATTTTGCAG
GTCAGTTAATTTCAGAAGCTGGCTTGAAAGGCTATCGTATCGGTGGCGTAGAAGTGTCAGAAAAGCATGC
AGGATTTATGATCAATGTCGCAGATGGAACGGCCAAAGACTACGAGGACTTGATCCAATCGGTTATCGAA
AAAGTCAAGGAACACTCAGGTATTACGCTTGAAAGAGAAGTCCGGATCTTGGGTGAAAGCCTATCGGTAG
CGAAGATGTATGCAGGTGGTTTTACTCCCTGCAAGAGGTAG
*GC% Content for gene: 45% [Found Using Genomics Content Calculator]
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Reference Sites:
-http://brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.158
-http://www.sigmaaldrich.com/catalog/search?interface=All&term=Nicotinamide%20Adenine%20Dinucleotide%20Phosphate&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286
-http://www.sigmaaldrich.com/catalog/search?interface=All&term=enolpyruvyl-UDP-N-acetylglucosamine&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286
-http://aac.asm.org/content/50/2/556#ref-list-1
-http://web.expasy.org/protparam/
-http://www.ch.embnet.org/software/TMPRED_form.html
-http://www.sciencebuddies.org/science-fair-projects/project_ideas/Genom_GC_Calculator.shtml